Hepatocyte Sirtuin 6 Protects against Atherosclerosis and Steatohepatitis by Regulating Lipid Homeostasis

Histone deacetylase Sirtuin 6 (SIRT6) regulates many biological processes. SIRT6 is known to regulate hepatic lipid metabolism and inhibit the development of nonalcoholic fatty liver disease (NAFLD). We aimed to investigate the role of hepatocyte SIRT6 in the development of atherosclerosis and further characterize the mechanism underlying SIRT6’s effect on NAFLD. Ldlr−/− mice overexpressing or lacking hepatocyte SIRT6 were fed a Western diet for 16 weeks. The role of hepatic SIRT6 in the development of nonalcoholic steatohepatitis (NASH), atherosclerosis, and obesity was investigated. We also investigated whether p53 participates in the pathogenesis of NAFLD in mice overexpressing hepatic SIRT6. Our data show that loss of hepatocyte SIRT6 aggravated the development of NAFLD, atherosclerosis, and obesity in Ldlr−/− mice, whereas adeno-associated virus (AAV)-mediated overexpression of human SIRT6 in the liver had opposite effects. Mechanistically, hepatocyte SIRT6 likely inhibited the development of NAFLD by inhibiting lipogenesis, lipid droplet formation, and p53 signaling. Hepatocyte SIRT6 also likely inhibited the development of atherosclerosis by inhibiting intestinal lipid absorption and hepatic VLDL secretion. Hepatic SIRT6 also increased energy expenditure. In conclusion, our data indicate that hepatocyte SIRT6 protects against atherosclerosis, NAFLD, and obesity by regulating lipid metabolism in the liver and intestine.


Introduction
Dysregulation of the epigenome drives aberrant transcriptional programs that promote disease onset and progression. Histone modification is a covalent post-translational modification that includes acetylation, methylation, phosphorylation, ubiquitylation, and sumoylation. Histone acetylation is regulated by adding or removing acetyl-CoA via histone acetyltransferases (HATs) and histone deacetylases (HDACs) in the lysine residues, respectively. Sirtuin 6 (SIRT6) is one of the NAD + -dependent sirtuins (SIRT1-7) that belong to class III HDACs. SIRT6, a stress-responsive protein deacetylase of both acetyl groups and long-chain fatty-acyl groups, is also a mono-ADP-ribosyltransferase that transfers ADP-ribose moieties to the lysine and arginine residues of protein substrates [1]. SIRT6 regulates cellular homeostasis by modulating DNA repair, telomere maintenance, and lipid and glucose metabolism. Therefore, it participates in a plethora of diseases such as aging, cancer, fatty liver disease, cardiovascular disease, obesity, diabetes, neurodegeneration, etc. [1,2].
The role of hepatic SIRT6 in lipid metabolism has been extensively investigated. Kim et al. showed that mice lacking hepatic Sirt6 accumulate hepatic triglyceride (TG) due to inhibition of genes involved in lipogenesis and glycolysis and induction of genes involved 2 of 14 in fatty acid oxidation (FAO) [3]. Naiman et al. showed that SIRT6 promotes hepatic FAO via PPARα [4]. Recently, Zhu et al. showed that mice lacking hepatic Sirt6 develop severe fatty livers when fed a Western diet and that Sirt6 inhibits lipogenesis by suppressing liver X receptor (LXR), carbohydrate response element-binding protein (ChREBP), and sterol regulatory element-binding protein 1 (SREBP1) [5]. Tao et al. reported that SIRT6 reduces LDL-C levels by inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9)mediated LDL receptor degradation [6] and that SIRT6 lowers hepatic cholesterol levels by repressing SREBP2 [7].
Despite significant research conducted on the role of hepatic SIRT6 in metabolic regulation, the role of hepatic SIRT6 in atherosclerosis or obesity has not been explored. In addition, the role of hepatic SIRT6 in NASH development is not fully understood. In this study, we used mice over-expressing or lacking hepatic SIRT6 to show that hepatic SIRT6 protects against Western diet-induced atherosclerosis, steatohepatitis, and obesity. The atheroprotective effect of hepatic SIRT6 is independent of LDLR. Furthermore, we reveal novel mechanisms contributing to hepatic SIRT6-mediated NASH development and atherogenesis.

Mice and Diets
C57BL/6J mice, albumin-cre (alb-cre) mice (stock # 003574), and Ldlr −/− mice (stock # 002207) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) on a C57BL/6J background. The Sirt6 fl/fl mice were described previously [3]. Sirt6 fl/fl mice were crossed with albumin-Cre mice to generate liver-specific Sirt6 −/− mice (Sirt6 Hep−/− ) and control (Sirt6 fl/fl ) mice. Sirt6 fl/fl mice and Ldlr −/− mice were cross-bred to generate Sirt6 fl/fl Ldlr −/− mice. A Western diet containing 21% fat/0.2% cholesterol (stock # TD.88137) was purchased from Envigo (Indianapolis, IN, USA). Unless otherwise stated, about two-month-old male mice were fed this special diet for four months and fasted for 5-6 h during the light cycle prior to anesthesia. All animal studies complied with the ARRIVE guidelines and were approved by the Institutional Animal Care and Use Committee at Northeast Ohio Medical University.

Real-Time PCR
Total RNA was isolated from the liver using Trizol (Invitrogen; cat #15596018). The genomic DNA was removed using the DNA-free™ Kit (Ambion; cat # AM1906). The cDNA was generated following the instructions of the TaqMan Reverse Transcription Kit (Applied Biosystems; Waltham, MA, USA; cat# N8080234). qPCR was performed using the PowerUp SYBR Green master mix (ThermoFisher Scientific; Waltham, MA, USA: cat# A25778) on a 7500 real-time PCR machine (Applied Biosystems; Waltham, MA, USA). Relative mRNA levels were quantified using the 2 −∆∆Ct method normalized to 36b4.

Liver Histology and Apoptosis Assays
Fresh liver samples were fixed in 10% formalin. Livers were dehydrated and frozensectioned by Cryostat (Leica CM1950; Deer Park, IL, USA) for Oil red O staining. Livers were paraffin-embedded and sectioned by microtome (Leica RM2235) for hematoxylin and eosin (H&E) staining, picrosirius red staining, or TUNEL assay. Staining of apoptotic nuclei was performed using a TUNEL assay kit (ab206386; Abcam; Cambridge, Cambridgeshire, UK).

Hepatic Lipids and Hydroxyproline
Hepatic total lipids were extracted from chloroform/methanol (2:1 v/v) using the Bligh and Dyer method, as described previously [13]. We then assessed triglyceride concentrations from the resulting emulsion using Infinity reagents (Thermo Fisher Scientific; Waltham, MA, USA). Free cholesterol and free fatty acid concentrations were measured according to the manufacturer's instructions (Fujifilm; Tokyo, Japan). We weighed a portion of fresh or frozen liver and used a kit from Cell Biolabs (STA675) to measure hepatic hydroxyproline levels. The result was expressed as µg/mg liver.

Intestinal Fat Absorption
Mice were fasted for at least 4 h, followed by an intravenous injection of tyloxapol (500 mg/kg). The mice were then gavaged with olive oil (15 µL/g body weight). Blood samples were drawn at various time points and triglyceride levels were quantified as described [14,15].

Intestinal Cholesterol Absorption
Mice were i.v. injected with 2.5 µCi 3 H-cholesterol in Intralipid (Sigma; St. Louis, MO, USA), followed by immediate gavaging with 1 µCi 14 C-cholesterol in median-chain triglycerides (MCT oil; Mead Johnson, Evansville, IN, USA). After 72 h, blood and tissue were collected. Plasma was collected to determine 3 H and 14 C activity. Cholesterol absorption was calculated as previously described [14,16].

Bile Acid Measurement
The total bile acids in the liver, intestine, and gallbladder were extracted in ethanol as described [17]. The bile acid concentration was quantified using the total bile acid assay kit from Diazyme (Poway, CA, USA; cat # DZ042AK01). We calculated the bile acid pool size based on the total amount of bile acids in the liver, intestine, and gallbladder.

Atherosclerotic Lesion Quantification
The whole aorta, including the ascending, thoracic, and abdominal segments, was isolated and cleaned under a microscope. The en face aortas and sectioned aortic roots were stained with Oil red O. The atherosclerotic plaque size was determined using ImageJ software from National Institutes of Health (Bethesda, MD, USA). The lesion was selected by the "Freehand tool", and the lesion areas in µm 2 were collected using "Control + M".

Body Composition and Energy Expenditure
We used EchoMRI™-700 (EchoMRI LLC, Houston, TX, USA) to measure the whole body fat and lean masses of the mice. The Comprehensive Lab Animal Monitor System (CLAMS) system was used to measure oxygen consumption and heat production, as described previously [18]. In brief, mice underwent an acclimation period, and 24 h measurement of energy expenditure was determined using an eight-chamber system. Each run included two genotypes with four mice per group.

Statistical Analysis
Statistical significance was analyzed using a student t-test or two-way ANOVA by Prism (GraphPad, Boston, MA, USA). All values were expressed as mean ± SEM. Differences were considered statistically significant at p < 0.05.

Hepatocyte SIRT6 Is Required for Protection against Western Diet-Induced Steatohepatitis
The role of hepatic SIRT6 in the development of diet-induced steatohepatitis has not been fully clarified to date. Hyperlipidemic Ldlr −/− mice develop severe liver steatosis, inflammation, obesity, and insulin resistance when fed a Western diet. For this reason, they have been used to study the development of NASH [19] and atherosclerosis. Therefore, we crossed Sirt6 fl/fl mice with Ldlr −/− mice to generate Sirt6 fl/fl Ldlr −/− mice, which were then i.v. injected with AAV8-TBG-Cre or AAV8-TBG-Null to generate Ldlr −/− mice with a hepatocyte-specific deletion of Sirt6 (Sirt6 Hep−/− Ldlr −/− ) and the control (Sirt6 fl/fl Ldlr −/− ) mice, respectively. These mice were fed a Western diet for 16 weeks. Compared to Sirt6 fl/fl Ldlr −/− mice, Sirt6 Hep−/− Ldlr −/− mice had a 77% reduction in hepatic SIRT6 protein levels ( Figure 1A,B). Sirt6 Hep−/− Ldlr −/− mice had increased plasma AST and ALT levels ( Figure 1C), and the ratio of liver to body weight ( Figure 1D). Oil red O staining ( Figure 1E Figure 1J) and hepatic hydroxyproline levels ( Figure 1K). Finally, Sirt6 Hep−/− Ldlr −/− mice had increased hepatic apoptosis ( Figure 1L,M). Thus, the data in Figure 1 demonstrate that hepatocyte SIRT6 is required for protection against diet-induced steatohepatitis.

Hepatic Expression of Human SIRT6 Prevents Western Diet-Induced Steatohepatitis
To address whether hepatic overexpression of SIRT6 regulates the development of NAFLD, we generated an AAV expressing human SIRT6 under the control of an albumin promoter (AAV8-ALB-hSIRT6). When fed a Western diet for 16 weeks, the hepatic expres- points in the graphs represent an individual mouse or a biological measurement. Statistical analysis was performed using a student t-test. * p < 0.05, ** p < 0.01.

Hepatic Expression of Human SIRT6 Prevents Western Diet-Induced Steatohepatitis
To address whether hepatic overexpression of SIRT6 regulates the development of NAFLD, we generated an AAV expressing human SIRT6 under the control of an albumin promoter (AAV8-ALB-hSIRT6). When fed a Western diet for 16 weeks, the hepatic expression of human SIRT6 in Ldlr −/− mice ( Figure 2A) reduced plasma AST and ALT levels (Figure 2B) and the ratio of liver to body weight ( Figure 2C). Hepatic overexpression of human SIRT6 also reduced hepatic neutral lipid accumulation ( Figure 2D,E), TG, FC, and FFA levels ( Figure 2F-H), fibrosis ( Figure 2I,J), and apoptosis ( Figure 2K,L). Thus, the data in Figures 1 and 2 demonstrate that hepatocyte SIRT6 protects against Western diet-induced steatohepatitis in Ldlr −/− mice. . All data are expressed as mean ± SEM. Data points in the graphs represent an individual mouse or a biological measurement. Statistical analysis was performed using a student t-test. * p < 0.05, ** p < 0.01.
(A,E,F,J) or two-way ANOVA (B-D, G-I). * p < 0.05, ** p < 0.01. . All data are expressed as mean ± SEM. Data points in the graphs represent an individual mouse or a biological measurement. Statistical analysis was performed using a student t-test. * p < 0.05, ** p < 0.01.

SIRT6 Reduces Hepatic Apoptosis and Lipid Levels Partly via p53
Apoptosis is believed to play an important role in NASH development by triggering hepatic inflammation [20]. The tumor suppressor protein p53 is involved in DNA repair and apoptosis [21] and the pathogenesis of NAFLD [22,23]. SIRT6 is shown to deacetylate lysine 382 of p53 [24]. Interestingly, overexpression of SIRT6 reduced p53 expression by >71% in Hepa1-6 cells (Supplementary Figure S1A,B) and C57BL/6 mice (Supplementary Figure  S2A,B). We then investigated whether p53 participates in SIRT6-mediated inhibition of NAFLD. Hepatic overexpression of human SIRT6 reduced the ratio of liver to body weight and hepatic TG and FFA levels in Western diet-fed C57BL/6 mice, which were blunted when p53 was overexpressed in the liver (Supplementary Figure S2C-F). Overexpression of human SIRT6 inhibited hepatic apoptosis in both control mice and p53-overpressing mice. p53 overexpression also normalized hepatic apoptosis in SIRT6-overexpressing mice (Supplementary Figure S2G-H). Thus, the data in Supplementary Figure S2 suggest that p53 plays a role in hepatic SIRT6-mediated inhibition of NAFLD.

Hepatic SIRT6 can Sufficiently Protect against the Development of Atherosclerosis in Ldlr −/− Mice
The role of hepatic SIRT6 in atherosclerosis has not been investigated to date. Loss of hepatocyte Sirt6 in Ldlr −/− mice raised plasma total cholesterol by 41% ( Figure 4A) and plasma triglyceride levels by 206% ( Figure 4B   quantified (H). All data are expressed as mean ± SEM. Data points in the graphs represent an individual mouse or a biological measurement. Statistical analysis was performed using a student t-test. * p < 0.05.
By contrast, total cholesterol ( Figure 5A) and triglyceride ( Figure 5B) levels in plasma from Ldlr −/− mice overexpressing hepatic SIRT6 were reduced by 37% and 39%, respectively. FPLC analysis showed that SIRT6 overexpression reduced VLDL-C, LDL-C (Figure 5C), and VLDL-TG ( Figure 5D). As a result, overexpression of hepatic SIRT6 reduced the lesion size of en face aortas ( Figure 5E,F) and aortic roots ( Figure 5G,H) by 32% and 35%, respectively. In summary, the data in Figures 4 and 5 demonstrate that hepatic SIRT6 sufficiently protects against diet-induced atherosclerosis. The lesions on the aortic roots were stained (G) and quantified (H). All data are expressed as mean ± SEM. Data points in the graphs represent an individual mouse or a biological measurement. Statistical analysis was performed using a student t-test. * p < 0.05.

Hepatic SIRT6 is Critical for Regulating Intestinal Cholesterol and Fat Absorption and VLDL Secretion
The inhibited development of atherosclerosis by SIRT6 in Ldlr −/− mice suggests that LDLR does participate in SIRT6-mediated suppression of atherosclerosis. In Ldlr −/− mice, loss of hepatocyte Sirt6 reduced hepatic mRNA levels of genes involved in cholesterol synthesis (HMG-CoA synthase (Hmgcs), HMG-CoA reductase (Hmgcr)), bile acid synthesis (cholesterol 7α-hydroxylase (Cyp7a1), sterol 12α-hydroxylase (Cyp8b1)), and VLDL secretion (microsomal triglyceride transfer protein (Mtp)) ( Figure 6A). There were only The lesions on the aortic roots were stained (G) and quantified (H). All data are expressed as mean ± SEM. Data points in the graphs represent an individual mouse or a biological measurement. Statistical analysis was performed using a student t-test. * p < 0.05.
In summary, the data in Figure 6 suggest that hepatic SIRT6 lowers plasma lipid levels by inhibiting intestinal cholesterol and fat absorption and hepatic VLDL secretion. Hepatic Cyp7a1 mRNA, protein, and Hmgcs mRNA levels were reduced in Ldlr −/− mice overexpressing hepatic SIRT6 ( Figure 6H-J). Interestingly, there were only minor changes in Hmgcr, Cyp8b1, Cyp27a1, Mtp, or Apob expression ( Figure 6H-J). Consistent with the inhibition of CYP7A1 expression, hepatic SIRT6 overexpression reduced bile acid pool size ( Figure 6K), cholesterol and fat absorption from the intestine ( Figure 6L,M), and VLDL secretion from the liver ( Figure 6N).
In summary, the data in Figure 6 suggest that hepatic SIRT6 lowers plasma lipid levels by inhibiting intestinal cholesterol and fat absorption and hepatic VLDL secretion.

Hepatic SIRT6 Is Required for Preventing Western Diet-Induced Obesity
Obesity is a major risk factor for metabolic disorders. Loss of hepatocyte Sirt6 in Ldlr −/− mice did not affect food intake (Supplementary Figure S3A), but increased body fat content by 156% ( Figure 7A). CLAMS studies showed that Sirt6 Hep−/− Ldlr −/− mice had reduced oxygen consumption ( Figure 7B,C) and heat production ( Figure 7D) during the day and night, whereas the respiratory exchange ratio (RER) was unchanged (Supplementary Figure S3B). At gene expression levels, loss of hepatocyte Sirt6 reduced uncoupled protein 1 (Ucp1) expression in brown adipose tissue (BAT) ( Figure 7E).

Hepatic SIRT6 Is Required for Preventing Western Diet-Induced Obesity
Obesity is a major risk factor for metabolic disorders. Loss of hepatocyte Sirt6 in Ldlr −/− mice did not affect food intake (Supplementary Figure S3A), but increased body fat content by 156% ( Figure 7A). CLAMS studies showed that Sirt6 Hep−/− Ldlr −/− mice had reduced oxygen consumption ( Figure 7B,C) and heat production ( Figure 7D) during the day and night, whereas the respiratory exchange ratio (RER) was unchanged (Supplementary Figure S3B). At gene expression levels, loss of hepatocyte Sirt6 reduced uncoupled protein 1 (Ucp1) expression in brown adipose tissue (BAT) ( Figure 7E).
Hepatic SIRT6 overexpression decreased fat content by 22% ( Figure 7F), increased oxygen consumption (Figure 7G,H) and heat production ( Figure 7I) as well as Ucp1 and Ucp2 expression in BAT ( Figure 7J). By contrast, there was no change in food intake or RER (Supplementary Figure S3C,D). Thus, hepatic SIRT6 is required to prevent diet-induced obesity by regulating energy expenditure.  injected with AAV8-ALB-Null or AAV8-ALB-hSIRT6 and fed a Western diet for 16 weeks (n = 8 per group). Fat content (F), oxygen consumption (G,H), heat production (I), and mRNA levels in BAT (J) were determined. All data are expressed as mean ± SEM. Data points in the graphs represent an individual mouse or a biological measurement. Statistical analysis was performed using a student t-test (A,E,F,J) or two-way ANOVA (B-D,G-I). * p < 0.05, ** p < 0.01.
Hepatic SIRT6 overexpression decreased fat content by 22% ( Figure 7F), increased oxygen consumption (Figure 7G,H) and heat production ( Figure 7I) as well as Ucp1 and Ucp2 expression in BAT ( Figure 7J). By contrast, there was no change in food intake or RER (Supplementary Figure S3C,D). Thus, hepatic SIRT6 is required to prevent diet-induced obesity by regulating energy expenditure.

Discussion
The role of hepatic SIRT6 in atherosclerosis or obesity has not been investigated before. In addition, the role of hepatic SIRT6 in the development of NAFLD has not been fully understood. In this work, we show that loss of hepatocyte SIRT6 aggravates Western dietinduced NAFLD, atherosclerosis, and obesity in Ldlr −/− mice. By contrast, AAV-mediated overexpression of human SIRT6 in the liver has opposite effects. Mechanistically, our data suggest that hepatocyte SIRT6 likely inhibits the development of NAFLD by suppressing de novo lipogenesis, lipid droplet formation, the p53 pathway, and inflammation. It also prevents the development of atherosclerosis by inhibiting intestinal fat and cholesterol absorption and hepatic VLDL secretion.
SIRT6 has been shown to lower plasma lipid levels by an unknown mechanism [25]. SIRT6 reportedly reduces LDL-C levels by inhibiting PCSK9-mediated LDLR degradation receptor degradation [6]. However, the loss or overexpression of hepatic SIRT6 markedly regulates plasma LDL-C levels and atherogenesis in Ldlr −/− mice, suggesting that LDLR does not mediate SIRT6's effects on plasma LDL-C levels. Our data show that hepatic SIRT6 inhibits cholesterol and fat absorption from the intestine and VLDL secretion from the liver. Furthermore, SIRT6 reduces CYP7A1 expression and bile acid pool size, which may contribute to changes in intestinal lipid absorption and hepatic VLDL secretion, since Cyp7a1 −/− mice displayed reduced cholesterol absorption [26] and over-expression of hepatic CYP7A1 increased VLDL secretion [27].
We also found that hepatic SIRT6 inhibits obesity. Although SIRT6 has been shown to regulate obesity [31], it has not been investigated whether hepatic SIRT6 regulates obesity. Our data show that hepatic SIRT6 reduces obesity by inducing UCP1 in BAT and energy expenditure. However, the precise mechanism remains elusive. Cyp7a1 −/− mice are resistant to diet-induced obesity via a yet-to-be-determined mechanism [27]. Our data show that SIRT6 inhibits CYP7A1 expression. Thus, hepatic SIRT6 likely inhibits diet-induced obesity by suppressing hepatic CYP7A1.
In summary, we identified hepatic SIRT6 as a key regulator of NAFLD, atherosclerosis, and obesity. Targeting hepatocyte SIRT6 may be useful for treating common metabolic disorders. One limitation of the current study is that we did not investigate atherosclerotic plaque composition, which will be further characterized in future work.